Three Gram-negative, non-pigmented, rod-shaped, facultatively aerobic bacterial strains, designated d8-1, d8-2 and IMCC1716, were isolated from a freshwater spring sample and a eutrophic freshwater pond. Based on characterization using a polyphasic approach, the three strains showed highly similar phenotypic, physiological and genetic characteristics. All of the strains harboured the nitrogenase gene , but nitrogen-fixing activities could not be detected in nitrogen-free culture media. The three strains shared 99.6–99.7 % 16S rRNA gene sequence similarity and showed 89–100 % DNA–DNA relatedness, suggesting that they represent a single genomic species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains d8-1, d8-2 and IMCC1716 formed a monophyletic branch in the periphery of the evolutionary radiation occupied by the genus . Their closest neighbours were Slu-05 (96.7–96.8 % similarity) and BS5-8 (96.3–96.6 %). The DNA–DNA relatedness of the novel strains to these two species of the genus was less than 70 %. The isolates could also be differentiated from recognized members of the genus on the basis of phenotypic and biochemical characteristics. It is evident, therefore, that the three strains represent a novel species of the genus , for which the name sp. nov. is proposed. The type strain is d8-1 (=LMG 24005=BCRC 17657).


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  1. Chen, W. M., Laevens, S., Lee, T. M., Coenye, T., de Vos, P., Mergeay, M. & Vandamme, P.(2001).Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int J Syst Evol Microbiol 51, 1729–1735.[CrossRef] [Google Scholar]
  2. Chen, W. M., Moulin, L., Bontemps, C., Vandamme, P., Bena, G. & Boivin-Masson, C.(2003). Legume symbiotic nitrogen fixation by beta-proteobacteria is widespread in nature. J Bacteriol 185, 7266–7272.[CrossRef] [Google Scholar]
  3. Chung, Y. C., Kobayashi, T., Kanai, H., Akiba, T. & Kudo, T.(1995). Purification and properties of extracellular amylase from the hyperthermophilic archaeon Thermococccus profundus DT5432. Appl Environ Microbiol 61, 1502–1506. [Google Scholar]
  4. Elliott, G. N., Chen, W. M., Chou, J. H., Wang, H. C., Sheu, S.-Y., Perin, L., Reis, V. M., Moulin, L., Simon, M. F. & other authors(2007).Burkholderia phymatum is a highly effective nitrogen-fixing symbiont of Mimosa spp. and fixes nitrogen ex planta. New Phytol 173, 168–180.[CrossRef] [Google Scholar]
  5. Embley, T. M. & Wait, R.(1994). Structural lipids of eubacteria. In Chemical Methods in Prokaryotic Systematics, pp. 121–161. Edited by M. Goodfellow & A. G. O'Donnell. Chichester: Wiley.
  6. Ezaki, T., Hashimoto, Y. & Yabuuchi, E.(1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[CrossRef] [Google Scholar]
  7. Felsenstein, J.(1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef] [Google Scholar]
  8. Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors)(1994).Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
  9. Hall, T. A.(1999).bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98. [Google Scholar]
  10. Hurek, T., Wagner, B. & Reinhold-Hurek, B.(1997). Identification of N2-fixing plant- and fungus-associated Azoarcus species by PCR based genomic fingerprints. Appl Environ Microbiol 63, 4331–4339. [Google Scholar]
  11. Kimura, M.(1983).The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.
  12. Kluge, A. G. & Farris, F. S.(1969). Quantitative phyletics and the evolution of anurans. Syst Zool 18, 1–32.[CrossRef] [Google Scholar]
  13. Kumar, S., Tamura, K. & Nei, M.(2004).mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.[CrossRef] [Google Scholar]
  14. Lanyi, B.(1987). Classical and rapid identification methods for medically important bacteria. Methods Microbiol 19, 1–67. [Google Scholar]
  15. MacFaddin, J. F.(2000).Biochemical Tests for the Identification of Medical Bacteria, 3rd edn. Baltimore, MD: Williams & Wilkins.
  16. Mesbah, M., Premachandran, U. & Whitman, W. B.(1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.[CrossRef] [Google Scholar]
  17. Moulin, L., Munive, A., Dreyfus, B. & Boivin-Masson, C.(2001). Nodulation of legumes by members of the β-subclass of Proteobacteria. Nature 411, 948–950.[CrossRef] [Google Scholar]
  18. Pot, B., Vandamme, P. & Kersters, K.(1994). Analysis of electrophoretic whole-organism protein fingerprints. In Chemical Methods in Prokaryotic Systematics, pp. 493–521. Edited by M. Goodfellow & A. G. O'Donnell. Chichester: Wiley.
  19. Powers, E. M.(1995). Efficacy of the Ryu nonstaining KOH technique for rapidly determining gram reactions of food-borne and waterborne bacteria and yeasts. Appl Environ Microbiol 61, 3756–3758. [Google Scholar]
  20. Quan, Z. X., Im, W. T. & Lee, S. T.(2006).Azonexus caeni sp. nov., a denitrifying bacterium isolated from sludge of a wastewater treatment plant. Int J Syst Evol Microbiol 56, 1043–1046.[CrossRef] [Google Scholar]
  21. Reinhold, B., Hurek, T., Niemann, E. G. & Fendrik, I.(1986). Close association of Azospirillum and diazotrophic rods with different root zones of Kallar grass. Appl Environ Microbiol 52, 520–526. [Google Scholar]
  22. Reinhold-Hurek, B. & Hurek, T.(2000). Reassessment of the taxonomic structure of the diazotrophic genus Azoarcus sensu lato and description of three new genera and new species, Azovibrio restrictus gen. nov., sp. nov., Azospira oryzae gen. nov., sp. nov. and Azonexus fungiphilus gen. nov., sp. nov. Int J Syst Evol Microbiol 50, 649–660.[CrossRef] [Google Scholar]
  23. Rosado, A. S., Duarte, G. F., Seldin, L. & Van Elsas, J. D.(1998). Genetic diversity of nifH gene sequences in Paenibacillus azotofixans strains and soil samples analyzed by denaturing gradient gel electrophoresis of PCR-amplified gene fragments. Appl Environ Microbiol 64, 2770–2779. [Google Scholar]
  24. Saitou, N. & Nei, M.(1987). The neighbor-joining method: a new method for constructing phylogenetic trees. Mol Biol Evol 4, 406–425. [Google Scholar]
  25. Sasser, M.(1990).Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.
  26. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors(1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef] [Google Scholar]
  27. Xie, C.-H. & Yokota, A.(2004). Phylogenetic analyses of the nitrogen-fixing genus Derxia. J Gen Appl Microbiol 50, 129–135.[CrossRef] [Google Scholar]
  28. Young, J. P. W.(1992). Phylogenetic classification of nitrogen-fixing organisms. In Biological Nitrogen Fixation, pp. 43–86. Edited by G. Stacey, R. H. Burris & H. J. Evans. New York: Chapman & Hall.
  29. Zani, S., Mellon, M. T., Collier, J. L. & Zehr, J. P.(2000). Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR. Appl Environ Microbiol 66, 3119–3124.[CrossRef] [Google Scholar]

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vol. , part 4, pp. 946 - 951

gene sequence-based phylogenetic tree generated by using the neighbour-joining method, showing the relationships between strains d8-1 , d8-2, IMCC 1716 and other nitrogen fixing bacteria.

Electron micrograph of cells of strain d8-1 .

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